This invention describes the synthesis and properties of a ovel class of chiral peptide nucleic acids (cPNAs) which hybridise strongly with complementary nucleic acids. As such they have potential as antigene and antisense agents and as tools in molecular biology.
Oligonucleotides are potentially useful for the regulation of genetic expression by binding with DNA or mRNA1. However, natural oligonucleotides are degraded by nucleases, consequently there is considerable interest in synthetic oligonudeotide analogues which are stable under physiological conditions. Recently, there has been interest in oligonucleotide analogues in which the sugar-phosphate backbone is replaced by a peptide chain2 after the success of the so-called Peptide Nucleic Acids (PNA)3, but more correctly referred to as Polyamide Nucleic Acids4.
The sugar phosphate backbone of a nucleic acid consists of a repeating unit of six atoms, configurationally and conformationally constrained by the Dribose or 2xe2x80x2-deoxy-D-ribose ring. If this could be replaced by a dipeptide unit the new backbone would be amenable to preparation by solid phase peptide synthesis. Molecular modelling by computer graphics suggested that a peptide chain consisting of an alternate sequence of a xe2x80x9cnucleo-amino acidxe2x80x9d derived from proline and a xe2x80x9cspacer amino acidxe2x80x9d, which could be any amino acid, should be a suitable structural analogue of the ribose phosphate backbone of nucleic acids as shown. 
This invention provides compounds of formula (I) 
where n is 1 or 2-200
B is a protected or unprotected heterocyclic base capable of Watson-Crick or of Hoogsteen pairing,
R is H, C1-C12 alkyl, C6-C12 aralkyl or C6-C12 heteroaryl which may carry one or more subsftuents preferably selected from hydroxyl, carboxyl, amine, amide, thiol, thioether or phenol,
X is OH or ORxe2x80x2 where Rxe2x80x2 is a protecting group or an activating group or a lipophilic group or an amino acid or amino amide or nucleoside,
Y is H or a protecting group or a lipophilic group or an amino acyl group or nudeoside.
When n is 1, these compounds are peptide nucleotide analogues. When n is 2 to about 30 these compounds are peptide oligonucleotides, which are synthesised as described below and can be s hybridised to ordinary oligo or polynucleotides. Typically the two strands are hybridised to one another in a 1:1 molar ratio by basespecific Watson-Crick base pairing.
B is a base capable of Watsonrick or of Hoogsteen pairing. This may be a naturally occurring nudeobase selected from A,C,G,T and U; or a base analogue that may be base specific or degenerate, e.g. by having the ability to base pair with both pyrimidines (T/C) or both purines (A/G) or universal, by forming base pairs with each of the natural bases without discrimination. Many such base analogues are known, e.g. hypoxanthene, 3-nitropyrrole, 5-nitroindole, and those cited by Lin and Brown5 and all are envisaged for use in the present invention.
The compounds of formula (I) contain proline of undefined stereochemistry. Although compounds with the trans -stereochemistry may have interesting properties, compounds with the cis-stereochemistry are preferred either with the Dcnfiguration as shown in (II) or the L-configuration shown in structure (III). In these compounds both stereoisomers of the xe2x80x9cspacer amino acidxe2x80x9d NHCHRCO are envisaged. 
Provided that it does not sterically hinder chain extension of hybridisation, the group R could have diverse structures. The group, however, can be chosen to confer desired hydrophobic, hydrophilic and/or electrostatic properties on the molecule. When the group R is other than H it generates a chiral centre and the two stereoisomers may allow for discrimination in the hybridisation of DNA and RNA. When the amino acid (xe2x80x94NHxe2x80x94CHRxe2x80x94CO) is a naturally occurring amino acid, then the amino acid should be readily and cheaply available as a building block for compounds of this invention. Any of the natural or unnatural xcex1-amino acids could be used e.g. glycine. or L- or D-serine or lysine. The nature of X can be varied from the negatively charged carboxylate ion (Xxe2x95x90O) to the incorporation of a positively charged lysine residue. Examples of the latter are provided in the experimental section and can be used to prevent aggregation and to assist hybridisation to the negatively charged oligonucleotides. Y will most commonly be H but could be any group which might be useful to improve the physical or biological properties of the material.
Any one of B, R, X and Y may include a signal moiety, which may be for example a radioisotope, an isotope detectable by mass spectrometry or NMR, a hapten, a fluorescent group or a component of a chemiluminescent or fluorescent or chromogenic enzyme system. The signal moiety may be joined to the peptide nudeotide analogue either directly or through a linker chain of up to 30 atoms as well known in the field.
In another aspect the invention provides a method of making the peptide nucleotide analogue of formula (I), comprising the steps of:
a) reacting an N-protected C-protected 4-hydroxy proline with a base selected from Nprotected thymine, N6-protected adenine, N4-protected cytosine, N2xe2x80x94O6-protected guanine and N3-protected uracil.
b) deprotecting the proline amino group of the product of a),
c) reacting the product of b) with an N-protected amino acid,
d) optionally removing protecting groups from the product of c).
In another aspect the invention provides a method of converting a peptide nucleotide analogue of formula (I) in which n is 1 into a peptide oligonucdeotide of formula (I) in which n is 2-200, comprising the steps of:
i) providing a support carrying primary amine groups,
ii) coupling an N-protected peptide nucleotide analogue of formula (I) to the support,
iii) removing the N-terminal protecting group,
iv) coupling an Nprotected nucdeotide analogue of formula (I) to the thus-derivatised support,
v) repeating steps iii) and iv) one or more times, and
vi) optionally removing the resulting peptide oligonucleotide from the support.
The invention also provides a compound of formula (IV) 
where R2 is H or a protecting group,
R3 is H or a protecting group compatible with R2, and
B is a protected or unprotected heterocyclic base.
The invention also provides a compound of formula (V) 
where R2 is diphenylmethyl, and
R3 is t-butoxycarbonyl.
The (2R,4R) (xe2x80x9ccis-Dxe2x80x9d)-proline was chosen since this is analogous to the stereochemistry of deoxyribonucleotides. The lack of negative charge on the peptide backbone would be expected to lead to a higher affinity for complementary oligonucleotide sequences in nudeic acids. Moreover these novel peptide nucleic adds can also be modified easily by using different xe2x80x9cspacer amino acidsxe2x80x9d to affect physical and biological properties such as solubility, cell permeability, etc. in order to achieve higher therapeutic activity. Such peptide nucleic acids should be stable to proteases since they contain substituted D-proline residues at alternate sites. Since coupling to secondary amino acids can be slow and inefficient, it was decided to use dipeptide building blocks in which the amino-acylprorine bond is formed in solution as in dipeptide (1). In the alternative arrangement, ire. prolyiamino acid, there is likely to be a serious problem of racemisation during the coupling if the amino acid is chiral, whereas such racemisation is not expected when the Gterminus of the activated fragment is proline because N-acylprolines can not racemise by the oxazolone mechanism6.
Because of the mild conditions used for the deprotection of the N-Fmoc group, the Fmoc/OtBu strategy in solid phase peptde synthesis is favoured over the classical Boc/OBzl strategy7. Furthermore, most machine synthesisers capable of handling small scale synthesis (50 xcexcmol or less) can accommodate only the Fmoc/OtBu strategy. For these reasons, it was decided to use the Fmoc instead of Boc as the N-protecting group.
There were two possible synthetic pathways to the target dipeptide (1), the two amino acids may be coupled first and the nucleobase attached later by the Mitsunobu reaction or the nucleobase may be incorporated before the peptide coupling. The first approach has the advantage of being a more convergent approach. However, a preliminary investigation suggested that it is not satisfactory because of the extensive cleavage of the Fmoc group during the Mitsunobu reaction. It also seemed likely that displacement of tosylate by a nucleobase would give similar premature cleavage of the Fmoc group since the reactions require basic conditions.
A temporary N-protecting group for the hydroxyproline was required, therefore, which is stable to the basic conditions of the Mitsunobu reaction but which can be removed, without disturbing the carboxyl protecting group, in order to allow coupling with Fmoc-glycine (or other amino acid) to give the Fmoc-dipeptide (1). As the carboxyl protecting group must be selectively removed in the presence of the Fmoc group at the end of the synthesis, an acidabile protecting group seemed appropriate. The combination of the acid labile Boc group and diphenylmethyl (Dpm) ester is ideal because introduction and cleavage of both groups are simple and high yielding. The Dpm ester is fully compatible with the N-Fmoc group and a selective cleavage of a Boc group in the presence of a diphenylmethyl ester is possible8.
Initial studies were undertaken with the commercially available trans-4-hydroxy-L-proline, which was protected as its N-Boc/Dpm ester derivative according to the method described by Tozuka and Takaya9. The crystalline derivative (2a) was obtained in greater than 80% yield in two steps.
The Mitsunobu reaction on (2a) with N3-benzoylthymine (BzT) gave the thymine derivative (3a), together with a less polar product, possibly the O2-isomer or the elimination product. Fortunately, the thymine derivative (3a) is crystalline and after column chromatography and one recrystallisation, the pure material was obtained in 51% yield.
Deprotection of the N-Boc group of the protected thymine derivative (3a) was accomplished with methanolic HCl. The resulting amine salt was reacted with Fmoc-glycne pentafluorophenyl ester in the presence of diisopropylethylamine (DIEA) to give the protected dipeptide (4a) in excellent yield. Treatment of (4a) with trifluoroacetic acid, either as a neat liquid or in the presence of phenol or anisole as a scavenger,10 at room temperature for a few hours led to the formation of roughly equal amounts of two products as shown by tic and hplc, which could not be separated by crystallisation. The unexpected product, which was more polar than the desired product, was identified as the debenzoylated thymine derivative (5a). Since protection of thymine at N3 was only required for selective alkylation at thymine-N1, the debenzoylated thymine derivative (5a) was suitable for oligomer synthesis. However, attempts to completely remove the benzoyl group by prolonged treatment with trifluoroacetic acid resulted in a complex mixture. HBr in acetic acid gave better results. Brief treatment of the mixture of products from trifluoroacetic acid cleavage with 10% HBr in acetic acid resulted in a complete cleavage of the benzoyl group as shown by hplc. The cleavage conditions have also been applied to the fully protected dipeptide (4a) without pretreatment with trifluoroacetic acid with equal success. The synthesis of Fmoc-dipeptide bearing thymine at 4-position in the CiS-L proline series is summarised in Scheme 1.
The protected cis- and trans-hydroxymproline (2b) and (2c) were required for the preparation of the trans- and cis-D-proline dipeptides bearing nucleobases. The reaction of N-Boc-cis-4-hydroxy-D-proline with diphenyldiazomethane gave the Dpm ester (2b) in 90% yield. Inversion of the 4-OH group in (2b) to give (2c) was effected by the Mitsunobu reaction. By this route, (2c) was prepared in multigram quantities from (2b) in excellent yield (90%, 2 steps) (Scheme 2). The specific rotation of the (2c) ([xcex1]D5+53.0, c=1.0, EtOH) when compared to that of the trans-L isomer ([xcex1]D25xe2x88x9254.3, c=1.0, EtOH) indicated that inversion was essentially complete.
The Mitsunobu reaction on the diastereomers, (2b) and (2c), with N3-benzoylthymine on a 20 mmol scale gave the products (3b) and (3c) in 33 and 36% yield respectively. The Boc group in (3b) and (3c) was removed with methanolic HCl and the products treated with Fmoc-glycine pentafluorophenyl ester to give the protected dipeptides (4b) and (4c). After treatment with 10% HBr in acetic acid the Fmoc-dipeptide acids (5b) and (5c) were obtained with concomitant cleavage of the N3-benzoyl group. The intermediate protected dipepfide (4b) and the final product (Sb) were not crystallised as readily as their diastereomers (4c) and (5c). However, the purity of the crude Fmoc-dipeptide (5b) and (5c) was proved to be satisfactory by hplc.
The Fmoc-dipeptide (6a), (5b) and (5c) were prepared in gramquantities for solid phase synthesis. Pentafluorophenyl esters of the diastereomeric thymine dipeptides (6a), (6b) and (6c) were all prepared by reactions of the free acids with pentafluorophenol in the presence of DCCl in dichloromethane.11 These active esters were crystalline solids which were stable enough to permit purification by silica gel column chromatography and could be stored for several months at xe2x88x9220xc2x0 C. without apparent decomposition according to 1H nmr.
Binding studies between the 10 mers derived from coupling of (6a), (6b) and (6c), and poly(dA), showed that the oligomer derived from (6c) binds most strongly. The Cis-D proline series was selected therefore for further investigation. The protected cis-hydroxy-D-proline (2b) was converted into the crystalline trans-D-tosylate (7) in 68% yield by a Mitsunobu reaction with methyl p-toluenesulfonate in the presence of triphenylphosphine and DEAD, according to the method of Peterson and Vince.12 Reactions of (7) and N6-benzoyladenine in the presence of K2CO3 and a catalytic amount of 18crown-6 in DMF afforded the N9-isomer of Boc-D-Pro(cis-4-BzA)-ODpm (8) in 42% yield. However, on scaling up, a small amount of another isomer (xcx9c5%) was also isolated. This was probably the N7-isomer according to the upfield 13C chemical shift of adenine C5 (115.0 and 114.6 ppm, rotamers) relative to the major product (123.4 ppm)13.
Deprotection of the Boc group in (8) was first attempted by methanolic HCl as described previously for the thymine derivatives, however, less selectivity was achieved. However, p-toluenesulfonic acid in acetonitrile, which has been successfully applied to deprotect the N-Boc group during the synthesis of cephalosporin derivatives,14 cleanly removed the Boc group without cleaving the Dpm ester. The product was reacted with Fmoc-glycine pentafluorophenyl ester to gave the Fmoc-dipeptide diphenylmethyl ester (9) in 85% yield. Deprotection of the Dpm ester with trifluoroacetic acid in the presence of anisole gave the free acid which was directly converted into the pentafluorophenyl ester (10) by reacting with pentafluorophenol in the presence of DCCl. The N6-benzoyl group on adenine remained intact throughout the reaction sequence. Attempted purification of the highly polar pentafluorophenyl ester (10) by column chromatography found only limited success. However, the crude product after trituration and washing with hexane, was shown by 1H nmr to contain approximately 10% of dicyclohexylurea (DCU) as the only contaminant, and was used successfully for solid phase peptide synthesis.
Reaction of the trans-D-tosylate (7) with N4-benzoylcytosine in the presence of K2CO3/18-crown-6 in DMF gave the desired N1-isomer, Boc-D-Pro(cis-4-N1-BzC)-ODpm, (11) in 25% yield along with the less polar O2-isomer in 41% yield, which could be readily separated by chromatography on silica gel. The identity of the two isomers was further confirmed by the characteristic downfield shift of the 13C resonance of C4, of the O2-isomer compared to the NN1-isomer. Since N4-benzoylcytosine was shown to be partially hydrolysed in hot 85% acetic acid to give uracil,15 the stability of this group towards acids was tested before attempting deprotection of the Boc group or the diphenylmethyl ester. The Boc-protected amino acid (11) was treated with trifluoroacetic acid in the presence of anisole for 2 h. 1H nmr of the product showed that the Boc and Dpm groups were completely removed whereas the benzoyl group was stable thus demonstrating that the deprotection conditions were satisfactory. Removal of the Boc group of (11) and reaction of the product with Fmoc-glycine pentafluorophenyl ester as described for the adenine analogue gave the protected cytosine dipeptide (12) in 70% overall yield.
The benzoylcytosine dipeptide and its pentafluorophenyl ester (13) were synthesised in essentially the same way as the thymine and adenine analogues.
The Mitsunobu reaction between N2-isobutyryl-O6(4xe2x80x2-nitrophenylethyl)guaninel6 and the protected trans-4-hydroxy-D-proline (2c) gave the required product, but, could not be isolated free from diethyl hydrazinedicarboxylate. Treatment with DBU in pyridine to remove the O6-nitrophenylethyl protecting group followed by column chromatography, however, gave the pure N9-substfduted isobutyrylguanine derivative (14) as a white solid in 43% overall yield. Removal of the Boc group and reaction of the product with Fmoc-glycine pentafluorophenyl ester gave the protected guanine dipeptide (15) in 52% yield. Removal of the carboxyl protecting group and reaction of the product with pentafluorophenol and DCCl gave the isobutyrylguanine dipeptide and its pentafluorophenyl ester (16).
A model synthesis was first carried out manually on the trans-D-proline analogue. The target was a T10 cPNA: H-[Gly-D-Pro(trans-4-T)]10-Lys-NH2. The lysine amide was included at the C-terminus to prevent self-aggregation and to increase water solubility. An acid-labile dimethoxybenzhydrylamine Novasyn-TGR resin was chosen as the solid support since cleavage with trifluoroacetic acid leads directly to the peptide amide. The polyethyleneglycol matrix also improves the swelling properties of the resin and allows better access of the reagents to the growing peptide chain. The first Wysine residue was introduced by coupling with Fmoc-Lys(Boc)OPfp in the presence of HOBt. The peptide was synthesised from Fmoc-Gly-D-Pro(trans-4-T)OH (5b), in the presence of HBTU/DIEA according to the standard protocol for Fmoc-solid phase synthesis.17 The efficiency of the coupling reactions, which was followed quantitatively after deprotection of the Fmoc group by measuring the absorbance of the dibenzofulvene-piperidine adduct (xcex5264=18000) liberated during deprotection, was not as good as expected despite the use of a large excess of reagents and prolonged reaction times.
Much better coupling was obtained by first converting (5b) into the pentafluorophenyl (Pfp) ester (6b) with DCCl and pentafluoro-phenol, and then performing the coupling in the presence of HOBt A possible reason for the low yield in the case of HBTU activation was that the activated monomer may have been lost by cyclisation to a diketopiperazine derivative. This is a very facile reaction for the active esters of protected or unprotected dipeptides which contain proline at the C-terrnini, especially in the presence of a base, for example, Z-Gly-Pro-ONp, is known to spontaneously cydise under basic conditions.18 The pentafluorophenyl ester is less reactive than the O-acylisourea or HOBt ester formed during HBTU activation and the coupling does not require basic conditions, which probably explains the improved coupling.
The decathymine chiral peptide nucleic acids with different stereochemistry at proline (trans-D, cis-D and cis-L), i.e. H-[Gly-D-Pro(trans-4-T)]10-Lys-NH2, H-[Gly-D-Pro(cis-4-T)]10-Lys-NH2 and H-[Gly-L-Pro(cis-4-T)]10-Lys-NH2 were successfully prepared by successive coupling of the corresponding dipeptide pentafluorophenyl esters according to the protocol shown in Scheme 3 on 5 xcexcmol scales. The syntheses were accomplished rapidly and efficiently. Total amounts of activated dipeptides required for each 5 xcexcmol synthesis of a decamer were approximately 150 mg. The chiral peptide nucleic acids were released from the resin and purified according to the standard protocol. In each case, analytical hplc of the crude products showed that they were 90-95% pure.
The peptides were purified by reverse phase hplc and their identity confirmed by electrospray mass spectrometry (Table 1). Interestingly, these higher oligomers showed an ability to form adducts with alkali metal ions especially potassium in the mass spectrometer, as evidenced by the presence of mass peaks at M+39n, where n is an integral number, in addition to the expected molecular ion peak. In some cases, these potassium ion adducts appeared as the major peaks in the mass spectra. All the T10 chiral peptide nucleic acids were sufficiently soluble in water for biological studies ( greater than 1 mg/mL at room temperature), although the trans-D-nanalogue was considerably more soluble than the other two.
Next the incorporation of different nucleobases into the chiral peptide nucleic acids was explored. The mixed adenine-thymine peptide nucleic acids of the transe and cis-L series were synthesised from the pentafluorophenyl esters without difficulty. However, attempts to remove the nucleobase protecting group (in this case, benzoyl) by treatment with aqueous ammonia under various conditions resulted in degradation of the peptide as shown by hplc. It seemed unlikely that the degradation resulted from direct hydrolysis or ammonolysis of the peptide bond, since the Gly-Pro and Pro-Gly bonds are stable to hot aqueous ammonia. Hplc and electrospray mass spectral analysis of the degradation products showed that they are the dipeptides Gly-Pro (or Pro-Gly) with the nucleobases remaining attached. This suggested that the degradation was probably caused by intramolecular attack by the amino group of the N-terminal glycine on the amide carbonyl of the next residue to release the bicyclic diketopiperazine, which could undergo further hydrolytic ring opening under the deprotection conditions to form the corresponding dipeptide observed in the mass spectrum. The process would be repeated until the entire peptide chain was degraded.
Understanding the mechanism of degradation made it possible to avoid this serious side reaction by modifying the N-terminus of the peptide nucleic acid in a way that would diminish the nucleophilicity of the amino groups. It was therefore decided to find another protecting group which could be removed under conditions compatible with the peptide, preferably without introducing additional steps. The Boc group was used as it is labile under the conditions for peptide cleavage from the resin but stable under the conditions necessary to deprotect the nucleobases on the solid support. This protection-deprotection scheme was tested by synthesizing two mixed A-T sequences H-[Gly-L-Pro(cis-4-T)]2-[Gly-L-Pro(cis-4-A)-Gly-Prob(cis-4-T)]2-Lys-NH2 and H-[Gly-L-Pro(cis-4-T)]6-[Gly-L-Pro(cis-4-A)-Gly-L-Pro(cis-4-T)]Lys-NH2The fully protected peptides were assembled on the solid support as usual and after the final removal of the N-Fmoc group, the free N-termini were capped with di-t-butyl dicarbonate (Boc2O) in the presence of DIEA in DMF. A qualitative ninhydrn test indicated that the coupling was essentially complete. After flushing the reaction vessels with DMF, the resins were treated with 1:1 ethylenediamine-ethanol at room temperature overnight. This deprotection reagent has been used as a milder alternative to aqueous ammonia for the base labile methylphosphonate oligonucleotides.19 The reagent was chosen here because the reaction could be carried out at room temperature and in the same vessel used for the peptide synthesis. Another advantage is that the resin swells better in this non-aqueous mediumxe2x80x94swelling properties of the solid support are crucial for solid phase reactions. The relatively non-volatile ethylenediamine and benzamide derivative from the cleavage reactions were easily removed by flushing the reaction vessels with DMF. Final cleavage and purification were carried out according to the standard method. Reverse phase hplc analysis of the completely deprotected peptides showed dean single products in each case. The identity of the products was confirmed by mass spectrometry {H-[Gly-L-Pro(cis-4-T)]2-[Gly-L-Pro(cis-4-A)-Gly-L-Pro(cis-4-T)]2-Lys-NH2: Mr calcd. 1832.84, found 1832.40xc2x10.10; H-[Gly-L-Pro(cis-4-T)]6-[Gly-L-Pro(cis-4-A)-Gly-L-Pro(cis-4-T)]2-Lys-NH2: Mr calcd. 2945.92, found 2945.32xc2x114}.
The synthesis of chiral peptide nucleic acids incorporating all four natural nucleobases was undertaken next. The first model sequence synthesised was the tetramer H-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-A)-Lys-NH2. All the coupling was carried out under the conditions described for the oligothymine peptide nucleic acids. The coupling efficiency was monitored by measuring the absorbance of dibenzofulvene-piperidine adduct from the deprotection step and showed that the guanine and cytosine could be introduced efficiently ( greater than 90% coupling yield, single coupling). The N-terrninus of the resin bound peptide was then capped with the Boc group after removal of the last N-Fmoc group and then the resin was treated with concentrated aqueous ammonia-dioxane 1:1 at 55xc2x0 C. overnight to remove the nucleobase protecting groups. Ethylenediamine was avoided in this instance because it had been shown to cause modification of the cytosine residue in oligonudeotides by displacement of the exocyclic amino group with the aminoethylamino group.2 Final deprotection of the Boc group and cleavage from the solid support was carried out according to the standard protocol. Reverse phase hplc analysis revealed a single major product which was shown to be the desired product by electrospray mass spectrometry {H-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-A)-Lys-NH2: Mr calcd. 1276.54, found 1277.00xc2x10.07}.
A decamer mixed-base peptide nucleic acid, H-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-T)Gly-D-Pro(cis-4-T)Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4C)-Gly-D-Pro(cis-4-T)-Lys-NH2, was also synthesised by the standard protocol giving the product (Mr calcd. 2973.15, found 2974.80xc2x10.35) in good yield and purity as judged by reverse phase hplc. The reverse phase hplc chromatogram and electrospray mass spectrum of the decamer are shown in FIG. 1 and FIG. 2.
Hybridization Studies
The deca-thymine glycyl-proline peptide nucleic acids with cis-L, trans-D and cis-D stereochemistry, and C-terminal L-lysinamide were mixed in 1:1 ratio with poly(rA) and poly(dA) in the presence of 150 mM NaCl and sodium phosphate buffer (10 mM Na+, pH 7.0) and the Tmdetermined (FIG. 3). The cis-D- and cis-L-PNAs, but not the trans-D-PNA showed well defined single-transition mewling curves with both poly(rA) and poly(dA). The magnitudes of absorbance change were of the order of 30-40%. The Tm were as shown in Table 2.
The slightly higher Tm for the chiral peptide nucleic acid.poly(rA) complexes suggests that they are slightly more stable than the peptide nudeic acid.poly(dA) complexes. Although both cis-D and cis-L analogues gave melting curves with poly(dA) and poly(rA), only the cis-D analogue gave a welldefined melting curve with (dA)10, with Tm=61xc2x0 C., under the same conditions. The cis-L analogue gave a broad melting curve with a Tm near room temperature. These results suggest that there is a stronger interaction between the cis-D analogue, which possessed the same absolute stereochemistry as natural oligonucleotides.
Further investigation of the nature of the cis-D peptide nucleic acid-oligonucleotide interaction was undertaken by determining the melting curve with (dT)10. The 1:1 mixture with (dT)10 showed no significant increase in absorbance at 260 nm on heating whereas the 1:1 mixture with (DA)10 showed strong hyperchromicity (ca. 20%) suggesting that the binding is probably specific for A.T pairs, presumably by Watson-Crick base pairing.
In order to determine the stoichiometry of the peptide nucleic acid-nucleic acid complexes, a titration experiment between cis-D-stereomer and poly(rA) was investigated. A well-defined mixing curve was obtained with minima at 1:1 ratio of peptide nucleic to nucleic acid in sodium phosphate buffer (10mM Na+, pH 7.0) suggesting a 1:1 stoichiontetry (FIG. 4). A similar titration experiment with (dA)10 under the same conditions gave essentially the same result.
All four bases found in DNA were introduced into the glycylproline building units with the cis-D configuration and from these mixed cPNAs containing all four nucleobases have been made. Sequence ID No. 1, GTAGATCACT, capped at its C-terminus with L-lysinamide was synthesised, and its binding properties with oligonucleotides investigated. Since it is important to determine the preferred orientation of binding of these novel cPNAs to oligonudeotides, both of the possible complementary oligonucleotides were prepared, i.e. 5xe2x80x2-CATCTAGTGA-3xe2x80x2, Sequence ID No. 2, and 5xe2x80x2-AGTGATCTAC-3xe2x80x2 Sequence ID No. 3, and hybridised with the chiral PNA. Their Tm values were 47xc2x0 C. and 43xc2x0 C. respectively indicating that the N-terninus of the cPNA preferentially binds to the 5xe2x80x2-terminus of the oligonucleotide, and the C-terminus to the 3xe2x80x2-terminus of the oligonucleotide. This is known as the antiparallel mode of binding, but it is seen that the stability of the alternative parallel binding complex is only slightly less stable.
Following the promising results obtained on the binding studies of the cPNA with complementary oligonucleotides by Tm measurement, a 1H NMR experiment was performed on the mixed sequence decamer, H-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4A)-Gly-D-Pro(cis-4-G)Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4T)-LysNH2, and its complementary oligonucleotides, both in parallel and antiparallel fashion. Unfortunately upon mixing the two components at the mmolar concentration required, a white precipitate formed immediately and no NMR signals could be observed apart from those of excess starting material. It is clear that even though the cPNA and the oligonucleotide alone are freely soluble in water, the complex formed between them is not at the high concentration required. In an attempt to overcome this problem it was decided to synthesise a cPNA analogue, containing a hydrophilic spacer amino acidxe2x80x94namely serinexe2x80x94in place of the glycine spacer in the backbone while retaining the cis-D-configuration of the proline moiety. As there are two enantiomers of serine and our preliminary molecular model suggested that the stereochemistry of the spacer amino acid may have considerable effects on the binding strength of the resulting diastereomeric cPNAs with A- and B-forms of DNA, both enantiomers of serine were studied. Thus the target molecules were diastereomeric thymine-decamers H-[L-Ser-D-Pro(cis-4-T)]10LysNH2 (xe2x80x9cLD-ST10xe2x80x9d) and H-[D-Ser-D-Pro(cis-4-T)]10LysNH2 (xe2x80x9cDD-ST10xe2x80x9d).
The required Fmoc-protected dipeptide diastereoisomeric synthons (17) were synthesised from Boc-D-Pro(cis-4-N3BzT)-Odpm (3c) in an analogous manner to the glycylproline analogue. The serine hydroxyl side chain was protected as a t-butyl ether as in the traditional Fmoc-OtBu orthogonal protection scheme. The Boc- group in the starting material was removed by p-TsOH in acetoninle as described previously, the amine tosylate was then reacted with Fmoc-Ser(OtBu)-OH in the presence of DCCl and HOBt in MeCN/DMF, after neutralisation with DIEA. Both enantiomers of serine gave similar yields (70-90%) of the desired protected dipeptides, Fmoc-L-Ser(OtBu)-D-Pr.(cis-4-BzT)-ODpm and Fmoc-D-Ser(OtBu)-D-Pro-(cis-4-BzT)-ODpm, which were isolated as white amorphous solids after column chromatography and were characterised by 1H, 13C NMR and APCIMS. Deprotection of the ODpm ester of Fmoc-L-Ser(OtBu)-D-Pro-(cis-4-BzT)-ODpm was found to be problematic since catalytic transfer hydrogenolysis using different hydrogen donors including ammonium formate, formic acid and cyclohexene and catalystsxe2x80x94Pd black, 10% Pd/C, and freshly prepared 5% Pd/BaSO4, gave unsatisfactory results. Aadic conditions were an alterative, if selectivity between the Dpm ester and t-Bu ether could be achieved. Various combinations were attempted, but 4M HCl in dioxane appeared to give the best result. The deprotected material, consisting a mixture of the desired Fmoc-dipeptide acid and some debutylated product was subjected to a reaction with pentafluorophenol/DCCl to give the final active ester of the Fmoc-dipeptide, which could be purified by column chromatography on silica gel (Scheme 4). The final products were obtained in a pure form and were characterised by 1H NMR and APCI-MS. It should be noted that the benzoyl protecting group on the thymine ring remained intact throughout the reaction sequence but this should not be a problem since this benzoyl group is removed readily upon treatment with 20% piperidine in DMF during deprotection of the Fmoc group in the solid phase synthesis step.
Solid phase synthesis of the two cPNA decamers, LD-ST10 and DD-ST10, were carried out according to our standard protocol on a 5 xcexcmol scale and the efficiency of each coupling step was monitored by measuring the absorbance of the dibenzofulvene-piperidine adduct released from deprotection of the Fmoc group and this showed that the coupling proceeded efficiently (95-100%). Both cPNA diastereomers could be purified to give the pure 10-mers by HPLC which gave correct masses by ESI-MS (3266, Mxe2x88x92H+K, identical spectra for both isomers). The purified cPNAs, LD-ST10 and DD-ST10, were obtained in 28 and 16% yield respectively. Both of the serine-containing cPNAs were freely soluble in water at a concentration of 2 mM.
A 1H NMR study of a mixture of DD-ST10 and dA10 was attempted. Initially the 1H NMR spectra of both components were recorded separately (at a concentration of 0.53 mM for the DD-ST10 and 0.67 mM for the dA10 in 10% D2O in H2O), which showed the expected resonances. On addition of 20 mol % of dA10 (as a concentrated aqueous solution) to a solution of 0.5 mM of DD-ST10 in 10% D2Oxe2x80x94H2O, an immediate precipitation occurred, and it was clear that a structure of the complex in solution could not be determined.